MXPA97003274A - Method of processing of a depressed current of the processing of qu - Google Patents

Abstract

The present invention relates to a method for processing a waste stream of cheese processing, characterized in that it comprises: i) contacting a waste cheese processing stream with a solvent and extracting a sialyl oligosaccharide with the solvent; a sialyl oligosaccharide containing solvent from the waste cheese processing stream, and iii) isolating a sialyl oligosaccharide from the solvent containing sialyl oligosaccharide.

Description

PROCESSING METHOD OF A CHEESE PROCESSING DISPOSAL CURRENT DESCRIPTION OF THE INVENTION This invention relates to a method of processing a waste stream from cheese processing. Whey is a main byproduct of "a cheese making, which, for environmental reasons", presents a difficult residual waste problem. The liquid is typically composed of about 5% by weight of lactose, 1% by weight of protein and about 0.5% by weight of salts, where the rest of the mixture is water. Although the protein component can often be recovered by ultrafiltration and therefore used in food products, the lactose component has hitherto been of little value. Even when countries that make major cheese continue to explore methods for disposing of and using fluid whey, the magnitude of the waste disposal problem requires that improved disposal methods be developed. In 1993 it was calculated by the Dairy Products Annual, USDA that more than 62.6 billion kilograms of serum fluid were produced only in the United States. Conventional waste methods for fluid serum include using it as a fertilizer, using it as liquid animal feed, using it in food products, drying and storage. Thomas in the U.S. 4,001,198 reports a method for recovering nutrients from cheese whey by multiple ultrafiltration stages, in which proteins, lactose and other small molecular weight molecules are successfully eliminated. The remaining permeable material is then oxygenated to decrease biological oxygen and chemical oxygen demands, such that the permeable material can be stored safely. Chambers et al. in the U.S. 4,547,386 reports the preparation of animal feed blocks from whey, in which the whey is concentrated at a solids content of at least 45%, followed by the addition of a divalent cation, to promote the gelation of the structure. Melnychyn in the U.S. 4,968,521 reports the use of fluid milk byproducts as an extraction solvent for the vegetable raw material in the production of a human food or new animal feed. Armstronq in the U.S. 4,617,861 reports the processing of cheese whey by separation of whey proteins, followed by fermentation of lactose to produce ethanol and fermentation of soluble materials. The protein fraction is used as a food source, e} ethanol as an industrial fuel and soluble fermentation matter as an animal feed supplement. Pederson, Jr. in the U.S. 4,202,909 reports the use of ultrafiltration to reduce the mineral content of cheese whey, making it easier to obtain relatively high purity lactose. Hariu et al. in the U.S. 4,971,701 and in 4,855,056 report the demineralization of cheese whey by means of a four chamber electrolytic cell. Harmon et al. in the U.S. 4,543,261 report the separation of soluble salts from low molecular weight, non-ionic organic compounds by passing a liquid right through a bed of a strongly acid, cation exchange type gel resin. Shimatani et al. in the U.S. 5,118,516 report the isolation of lactose containing sialic acid from whey, skim milk or deproteinized solution by (a) electrodialysis, or (b) ion exchange by a cation exchange resin and a strongly basic anion exchange resin, or (c) ) a combination of electrodialysis and ion exchange by the cation exchange resin and the strongly basic anion exchange resin to desalt the permeable material.

Shimatani et al. in the U.S. 5,270,462 report a manufacturing process of a composition containing a high concentration of sialic acids, adjusting the pH of the cheese whey to be acidic, contacting the whey with a cation exchanger, followed by concentration and desalting of the eluent. JP Kokai 01-168,693 reports the preparation of a sialic acid composition, subjecting milk, nonfat milk, butter or whey to ultrafiltration, fractionation from 20,000 to 500,000 Dalton units at a pH of 4.0 to 6.0, followed by a second ultrafiltration, fractionation from 1,000 to 10,000 Dalton units at a pH of 6.0 to 8.0 under 0.2 to 2.0 MPa, to remove impurities such as lactose. The residue is spray-dried or lyophilized. JP Kokai 03-143,351 reports the recovery of sialic acid from the oligosaccharide binding type from an alkaline cleaning waste liquid of the anion exchange resin formed in the desalination of the serum, by neutralization, ultrafiltration, reverse osmosis, desalination. , absorption of the sialic acid on a strongly basic type anion exchange resin, followed by elution, desalination and drying. JP Kokai 59-184,197 reports the manufacture of oligosaccharides linked to sialic acids, by desalting elasas containing sialyl oligosaccharide, passing the desalted solution through an anion exchange column, neutralizing the eluate and desalting the eluate by electrophoresis. Accordingly, the processing of waste streams from the cheese process by extraction of the valuable components by ion exchange chromatography have been reported. The ion exchange methods for removing the sialyl oligosaccharides from the waste stream from cheese processing experience the difficulty in isolating the sialyl oligosaccharide from the ion exchange column. Specifically, the absorbed sialyl oligosaccharide is eluted by treating the column with several column volumes of an aqueous salt solution such as NaCl and NaOAc. The result is an eluent of a very dilute solution of the sialyl oligosaccharide and a high salt concentration. To separate the sialyl oligosaccharide from the eluent, the eluent is subjected to desalting techniques such as reverse osmosis, which separates the sialyl oligosaccharide from the salt. However, reverse osmosis is a slow and energy intensive technique. Therefore, any isolation procedure, which can avoid the desalination stage by reverse osmosis would be advantageous.

Despite many creative methods of disposing of fluid waste, the increasingly increasing supply of cheese whey requires more effective waste disposal methods. The present invention solves this problem by providing a method of processing a waste cheese processing stream, which provides economical disposal of the material. Accordingly, an object of this invention is to provide a new method of processing the waste stream from cheese processing. A second object of this invention is to provide a second method of processing the cheese processing waste stream which is cheese whey. A third object of this invention is to provide a new method of processing a waste stream of cheese processing, which are solids obtained by drying cheese whey. A fourth object of this invention is to provide a new method of processing a waste stream from cheese processing, which is mother liquor obtained by the separation of lactose from cheese whey. A fifth object of this invention is to provide a new method of processing the cheese processing waste stream, which is lactose.

The objects of the present invention are provided by a method of processing the waste stream from cheese processing, which sequentially comprises: i) contacting a cheese waste stream with an anion exchange resin; ii) removing the anion exchange resin from the cheese processing waste stream and eluting the anion exchange resin with an aqueous solution of a lithium salt, to produce an eluent; iii) concentrating the eluent to form a solid of a lithium salt and a lithium salt of a cheese processing waste; and iv) washing the solid with an organic solvent, dissolving the lithium salt and leaving the lithium salt of a waste from processing the cheese as a solid. It has been found that the lithium salt of a cheese processing waste has little solubility in an organic solvent, while the lithium salts have high solubility in an organic solvent. Accordingly, the eluent can be "desalted" by a simple washing by separating it from the lithium salts from the lithium salts of the cheese processing waste with an organic solvent.

The process may furthermore consist in positively removing the charged materials from contacting the waste stream from the processing of the cheese with the anion exchange resin, followed by processing according to steps i) to iv). According to a second embodiment of the present invention, a cheese processing waste stream is treated, comprising sequentially: i) contacting a cheese processing waste stream with a solvent; ii) separating the solvent from the cheese processing waste stream; and iii) isolating an extract from a cheese processing waste stream. It has been found that a stream of cheese processing waste can be extracted with a solvent to obtain an extract from the waste stream of cheese processing. The cheese processing waste stream, which is processed according to the present invention, can be obtained from any waste stream generated during a cheese making process. For example, acid whey is generated by separating the solids when the skim milk is coagulated to form cotija cheese. The acid whey is characterized by a high content of lactic acid. When the cheese is prepared from whole milk, the remaining liquid is sweet whey, which can be further processed by evaporation to form dry whey powder. The sweet serum can also be dried, demineralize and evaporate to form a permeate of demineralized serum. The sweet serum can also be subjected to ultrafiltration to generate both a permeate of serum and a permeate concentrate of serum. The serum permeate can also be processed by crystallization of the lactose, to form both lactose and a mother liquor. The mother liquor resulting from the crystallization of lactose from a permeate of whey is known in the art as "Delac". Suitable cheese processing waste streams include colostrum, milk, milk powder, whole serum, demineralized serum permeate, regeneration stream of demineralized permeate, serum permeate, crystallized lactose, spray dried lactose, whey powder, edible lactose, lactose, refined lactose and lactose USP. Preferably, the aqueous mother liquor material resulting from the crystallization of the lactose (ie Delac) is used. The fluid cheese whey is typically used to produce a highly dispersible, non-hygroscopic powder. The fresh fluid serum is clarified by passing it through a clarifier of the elimination type of 1 or mud. The serum is separated to remove the graea, then concentrated in double or triple effect evaporators to a solids content of approximately 62% by weight. The solids can be removed by separation at room temperature, or more preferably, the concentrated whey is cooled before the solids are removed. When the waste stream from the processing of the cheese to be processed is the solids obtained from the drying of the whey, the solids may first be dissolved in water, preferably in an amount of about 1 to 620 g, preferably 50 to 200 g, more preferably about 100 g of solids per liter of water. The dissolution of the solids obtained from the drying of the cheese whey can be carried out at room temperature or at elevated temperatures to accelerate the dissolution process and increase the amount of dissolved solids. Preferably, temperatures of 20-80 ° C are adequate. Alternatively, solids can be processed directly by extraction with a solvent. Typically the cheese processing waste stream can be used in the present invention, without adjusting the pH. Accordingly, there is generally no need to change the pH of the material before it is processed according to the present invention. As such, this method can prevent the creation of even more waste, which could result if it were necessary to adjust the pH. However, if the pH of the cheese processing waste stream is not compatible with the present process, the pH can be brought within a pH range of 2-9 by the addition of an acid, such as hydrochloric acid, sulfuric acid, acetic acid, lactic acid or citric acid, at a pH of 2 to 10, preferably 3 to 9, more preferably 4 to 6, by the addition of a base such as sodium hydroxide, ammonium hydroxide and potassium hydroxide. Prior to treatment of the cheese processing waste stream with the anion exchange resin, the cheese processing waste stream of preference is first treated to remove whey proteins and other positively charged materials. The pretreatment to positively remove the charged materials, allows the processing of a greater amount of cheese processing waste stream on the anion exchange resin, before the anion exchange resin needs to be regenerated. Any technique known to those of ordinary skill in the art can be used to eliminate positively charged materials. For example, a suitable technique for causing the whey protein to be absorbed is by contacting a cation exchange resin, as described by J.N. DeWitt et al (Neth Milk Dairy J., 40: 41-56 (1986)) and J.S. Ayers et al (New Zealand J. Dairy Sci. &Tech., 21: 21-35 (1986)), as well as those processes described in JP 2-104246 and 2-138295. Suitable cation exchange resins can be prepared by conventional techniques known to those of ordinary skill in the art. For example, a suitable cation exchange resin can be produced from a mixture of a monofunctional and polyfunctional monomer polymerizable by radical emulsion polymerization techniques, then functionalized with acid groups such as carboxylic acid groups or sulfonic acid groups that exist protonated form. The degree of crosslinking in the cation exchange resin can be chosen, depending on the operating conditions of the cation exchange column. A highly crosslinked resin offers the advantage of durability and a high degree of mechanical integrity, however it experiences a decreased porosity and a drop in mass transfer. A low-crosslinked resin is more brittle and tends to expand by the absorption of the mobile phase. A suitable resin can have from 2 to 12% crosslinking, preferably 8% crosslinking. The particle size of the cation exchange resin is selected to allow efficient flow of the cheese processing waste stream, while still effectively removing the charged materials. A suitable particle size for a column of 30 x 18 cm is 100-200 mesh. Suitable cation exchange resins include, but are not limited to CM-Sephadex, SP-Sephadex, CM-Sepharose, S-Sepharose, CM-Cellulose, Cellulose Phosphate, Sulfoxyethyl-Cellulose, Amberlite, Dowex-50, Dowex HCR-S , Dowex Macroporous Resin, Duolit C433, SP Trisacryl Plus-M, SP Trisacryl Plus-LS, Oxycellulose, AG 50W-X2, AG50W-X4, AG50 -X8, AG 50W-X12, AG 50 -X16, AG MP-50 Resin, Bio-Rex 70.

Most preferably the suitable resins are DOWEXMR 50x8 (an aromatic sulfonic acid bonded to a crosslinked polystyrene resin from Dow Chemical) and the acidic resins AMBERLYSTMR-15, AMBERLITEMR IR-120 and AMBERLITEMR-200. The waste stream from the processing of the cheese may be contacted with the cation exchange resin in any suitable form, which would allow the whey proteins and other positively charged materials to be absorbed onto the cation exchange resin. Preferably, the cation exchange resin is loaded onto a column and the cheese processing waste stream is passed through the column to remove the whey proteins. An amount of cation exchange resin is selected to effect the removal of the positively charged materials and will vary greatly depending on the processing waste stream of the cheese being treated. Typically, when the waste stream is permeated with whey, the charge ratio of the cheese processing waste stream to the cation exchange resin may be 5 to 20, preferably 8-15, most preferably 9. to 12: 1 v / v. When the contact is made in a column, the cheese processing waste stream is preferably passed at a speed of from 1 to 70 cm / minute, preferably from 2 to 15 cm / minute, more preferably at a rate of 4.6 cm / minute. A suitable pressure can be selected to obtain the desired flow velocity. Typically a pressure of 0 to 7.03 kg / cm2. { from 0 to 100 PSIG) is selected. Suitable flow rates can also be obtained by applying a negative pressure to the elution end of the column and collecting the eluted material. A combination of both of the positive and negative pressure can also be used. The temperature used for contacting the cheese processing waste stream of the cation exchange resin is not particularly limited, as long as the temperature is not too high to cause decomposition of the components of the waste stream. Generally the ambient temperature of 17 to 25 ° C is used. Alternatively, positively charged materials can be removed by such techniques as electrophoresis, ultrafiltration, reverse osmosis or salt precipitation. After the optional treatment of the waste stream from the cheese processing to remove the positively charged materials, the waste stream processing the cheese is contacted with an anion exchange resin. Suitable anion exchange resins can be prepared by conventional techniques known to those of ordinary skill in the art. For example, a suitable anion exchange resin can be produced from a monofunctional and polyfunctional monomer mixture polymerizable by radical emulsion polymerization techniques, then functionalized with strongly basic groups such as quaternary ammonium groups. The degree of crosslinking in the anion exchange resin can be chosen, depending on the operating conditions of the anion exchange column. A suitable resin can have from 2 to 12% crosslinking, preferably 8% crosslinking.

The particle size of the anion exchange resin is selected to allow efficient flow of the cheese processing waste stream, while still effectively removing the negatively charged materials. A suitable particle size for a column of 30 x 18 cm is 100-200 mesh. Suitable anion exchange resins include, but are not limited to, DEAE Sephadex, QAE Sephadex, DEAE Sepharose, Q Sepharose, DEAE Sephacel, DEAE Cellulose, Ecteola Cellulose, PEI Cellulose, QAE Cellulose, Amberlite, Dowex 1-X2, Dowex 1-X4, Dowex 1-X8, Dowex 2-X8, Dowex Macroporous Resins, Dowex GR-2, DEAE Trisacryl Plus-M, DEAE Trisacryl Plus-LS, Amberlite LA-2, AG 1-X2, AG 1-X4, AG 1-X8, AG 2-X8, AG MP-1 Resin, AG 4-X4, AG 3-X4, Bio-Rex 5 and ALIQUAT-336 (Tricaprylmethylammonium chloride from Henkel Corp .). More preferably, the suitable anion exchange resins are D0WEXMR 1X8 (a methylbenzylammonium bonded to a crosslinked polystyrene resin from Dow Chemical) and strongly basic resins AMBERLYSTMRA-26, AMBERLITEMR IRA 400, AMBERLITEMR IRA 400, AMBER ITEMRIRA 416 and AMBERLITEMR IRA 910. The cheese processing waste stream may be in contact with the anion exchange resin in any suitable form, which would allow the negatively charged materials to be absorbed onto the anion exchange resin. Preferably, the anion exchange resin is loaded onto a column and the cheese processing waste stream is passed through the column to absorb the negatively charged materials on the resin. An amount of anion exchange resin is selected to affect the absorption of the negatively charged materials and will vary greatly depending on the processing waste stream of the cheese being treated. Typically, when the waste stream is permeated with whey, the charge ratio of this cheese processing waste stream to the anion exchange resin is from 5 to 200, preferably from 8-15, most preferably from 9 to 12: 1 v / v. When the contact is affected in a column, the waste stream of cheese processing is preferably passed at a rate of 1 to 70 cm / minute, preferably 2 to 15 cm / minute, more preferably at a speed 4.6 cm / minute. A suitable pressure can be selected to obtain the desired flow rate. Typically a pressure of 0 to 7.03 kg / cm2 (from 0 to 100 PSIG) is selected. Suitable flow rates can also be obtained by applying a negative pressure to the elution end of the column and collecting the eluent. A combination of both of the positive and negative pressure can also be used.

The temperature used for the contact of the cheese processing serum stream with the anion exchange resin is not particularly limited, as long as the temperature is not too high to cause the decomposition of the components of the waste stream. Generally the ambient temperature is used from 17 to 25 ° C. By contacting the eluent in the anion exchange resin, the negatively charged components of the cheese processing waste stream are absorbed onto the anion exchange resin. The materials absorbed on the anion exchange resin are negatively charged materials from a cheese processing waste, which includes but is not limited to sialyl oligosaccharides such as 3'-sialyl-lactose, 6'-sialyl-lactose and 6'-sialyl -lactosamine. It is the elimination of sialyl oligosaccharides, which provides the economic treatment and disposal of the waste stream from cheese processing. As previously mentioned, the serum of the fluid that is produced at a rate of approximately 62.6 billion kilograms annually only in the United States. The lack of any recognized value has caused this material to be discarded as animal feed, fertilizer or conventional waste disposal techniques such as buried or stored. It was also discovered that disposal of the cheese processing waste stream can be made economical by the removal of the sialyl oligosaccharides from the cheese processing waste stream. Sialyloligosaccharides, such as 3'-sialyl lactose, 6'-sialyl lactose and 6'-sialyl lactosamine are useful as bacterial antiadhesives, anti-infectives and as an additive for infant formula. The utility of the sialic acid containing the compositions was reported in the U.S. 5,270,462. Sialyl lactose is also reported to be useful in a method for the treatment of arthritis (U.S. 5,164,374). However, sialyl oligosaccharides are very expensive due to their low amount found in natural sources. 3'-Sialyl lactose, isolated from bovine colostrum, is sold by Sigma Chemical Company for $ 60.05 per milligram. The 6 'isomer, also isolated from bovine colostrum, is sold for $ 66.10 per milligram. It has been found that up to 6 grams of sialyl oligosaccharides can be obtained per kilogram of waste stream from cheese processing. Since the waste stream of cheese processing is currently of little or no commercial value, it can be obtained cheaply, as an industrial waste product. A kilogram of previously worthless material can be processed, to extract components worth more than $ 60,000. By doing so, "now" economically viable to dispose of the waste stream of cheese processing. The resulting liquid, after contact with the anion exchange resin, which contains mainly water and lactose, can be dried and discarded as animal feed, fertilizer or as a food supplement. The anion exchange resin is then purged from the sialyl oligosaccharide by elution with an aqueous solution of a suitable salt such as sodium acetate, ammonium acetate, sodium chloride, sodium bicarbonate, sodium formate, ammonium chloride or a lithium salt. such as lithium acetate, lithium bicarbonate, lithium sulfate, lithium formate, lithium perchlorate, lithium chloride and lithium bromide as an eluent. The purging of an anion exchange resin with an aqueous salt can be accomplished by conventional means known to those of ordinary skill in the art. The sialyl oligosaccharide can also be removed from the anion exchange resin with an aqueous alkaline solution, although the concentration of the aqueous alkali must be diluted sufficiently so as not to destroy the structure of the sialyl oligosaccharide. The proper desorption conditions can be determined by means of routine experimentation. When eluting with an aqueous solution of lithium salts, no desalination by reverse osmosis is not necessary. The complete eluent can be concentrated and dried, then the remaining solids washed with an organic solvent. The lithium salts are dissolved and the lithium salt of the sialyl oligosaccharide remains as a solid. Specifically, the lithium salts of 3'-sialyl-lactose, 6'-sialyl-lactose and 6'-sialyl-lactosamine have been found to have very low solubility in organic solvent. The lithium salts used in the eluent must be freely soluble in water and have high solubility in an organic solvent. In the context of the present invention, a high solubility in an organic solvent is >; 1 g of lithium salt per ml of organic solvent, preferably > 5 g / ml, more preferred > . 10 g / ml at the temperature at which the solids are being washed. Suitable lithium salts which have been found to be freely soluble in water and have a high solubility in organic solvents include, lithium acetate, lithium bicarbonate, lithium sulfate, lithium formate, lithium perchlorate, lithium chloride and Lithium bromide.

The organic solvent used to wash the concentrated eluyepte must dissolve the lithium salt that elutes, still has little solvation effect on the lithium salt of a sialyl oligosaccharide. In the context of the present invention, a low solvation effect on the lithium salt of a sialyl oligosaccharide is when the solubility of the lithium salt of the sialyl oligosaccharide is > 0.5 g per ml of organic solvent, preferably > 0.25 g / ml, more preferred > 0.1 g / ml at the temperature at which the solids are being washed. Suitable solvents include, but are not limited to acetone, methyl ethyl ketone, 3-pentanone, diethyl ether, t-butyl methyl ester, methanol, ethanol and a mixture thereof. The organic solvent preferably contains < 0.1% by weight, more preferred by < 0.01% by weight of water, more preferably the organic solvent is anhydrous. The use of an organic solvent containing high concentrations of water results in the dissolution of the lithium salts of sialyl oligosaccharide. The temperature of the organic solvent is not particularly limited, however preferably the organic solvent is at room temperature or lower, more preferably 0-5 ° C. Due to the high hygroscopicity of the lithium salts of the sialyl oligosaccharide, the washing of the solids is carried out under conventional conditions, which are known to those of ordinary skill in the art, to limit the absorption of atmospheric moisture. For example, such washing can be carried out under an inert atmosphere, in a drying box or using a Schlenk type apparatus. When purging the anion exchange resin, with an eluent, a suitable purge solution is 50 mM. The pH of the eluent is preferably adjusted to be from 4 to 9, more preferably from 5 to 5. Generally from 2 to 5, preferably from 4 volumes of the purge solution column, it is used to remove the sialyl oligosaccharides. of the anion exchange resin, preferably carried out at room temperature. Preferably, the lithium acetate is used to purge the anion exchange resin of the sialyl oligosaccharides. The sodium salt of the sialyl oligosaccharides can be obtained by conventional ion exchange techniques, known to those of ordinary skill in the art. When an eluent of part of the lithium salt is used to remove the sialyl oligosaccharides from the anion exchange resin, the eluent containing the sialyl oligosaccharides and the salt can be concentrated and desalted such as by subjecting the eluent to reverse osmosis to remove the salt of the sialiloligosaccharide. The reverse osmosis can be carried out through a membrane with a molecular weight exclusion of 100 to 700 Dalton units, preferably an exclusion of 400 Dalton units. The reverse osmosis is preferably carried out at a pressure of 21.0-112.49 kg / cm2 (300-1,600 psi), more preferably 28.12-42.18 kg / cm2 (400-600 psi), even more preferably at a pressure of 31.63 kg / cm2 (450 psi). After the salts have been removed by reverse osmosis, the resulting material can be concentrated to provide a solid material containing the sialyl oligosaccharides such as 3'-sialyl-lactose and 6'-sialyl-lactose, which can be recrystallized from of a mixture of water and organic solvents. Preferably the precipitating solvents are selected from the group of ethanol, acetone, methanol, isopropanol, diethyl ether, t-butyl methyl ether, ethyl acetate, hexane, tetrahydrofuran and water. In addition, the elution of the anion exchange column, which contains a mixture of sialyl oligosaccharides which include 3'-sialyl-lactose, 6'-sialyl-lactose and 6'-sialyl-lactosa, can be subjected to separation of the sialiloligosaccharides contained therein, by column chromatography on an anion exchange resin DOWEX 1 x 2, at a pH of 4 to 6 using a suitable salt buffer such as sodium acetate, ammonium acetate or a lithium salt such as lithium acetate, lithium perchlorate, lithium chloride and lithium bromide as an eluent. A solution of lithium acetate is preferred. Suitable anion exchange resins can be prepared by conventional techniques known to those of ordinary skill in the art as previously described. The degree of crosslinking in the anion exchange resin can be chosen, depending on the operating conditions of the anion exchange column. A suitable resin can have from 2 to 12% crosslinking, preferably 2% crosslinking. The particle size of the anion exchange resin is selected to allow efficient flow of the cheese processing waste stream, while still effectively affecting the chromatographic separation of the negatively charged materials. A suitable particle size for a 20 x 100 cm column is 200-400 mesh. Suitable anion exchange resins include, but are not limited to, DEAE Sephadex, QAE Sephadex, DEAE Sepharose, Q Sepharose, DEAE Sephacell, DEAE Cellulose, Ecteola Cellulose, PEI Cellulose, QAE Cellulose, Amberlite, Dowex 1-X2, Dowex 1- X4, Dowex 1-X8, Dowex 2-X8, Dowex Macroporous Resins, Dowex WGR-2, DEAE Trisacryl Plus-M, DEAE Trisacryl Plus-LS, Amberlite LA-2, AG 1-X2, AG 1-X4, AG 1 -X8, AG 2-X8, AG MP-1 Resin, AG 4-X4, AG 3-X4, Bio-Rex 5 and ALIQUAT-336 (tricaprylmethylammonium chloride from Henkel Corp.). The preferred resins are DOWEX 1X2 (a tri-methylbenzylammonium bonded to a crosslinked polystyrene resin from Dow Chemical) and the basic resins AMBERLYST and AMBERLITE. The mixture of the sialyl oligosaccharides to be separated is subjected to column chromatography on an anion exchange resin. An amount of the anion exchange resin is selected to affect the separation of the different sialyl oligosaccharides. Typically, the charge ratio of the sialyl oligosaccharides to the anion exchange resin is 0.1 to 5, preferably 0.2 to 4, most preferably 1 gram of material per liter of resin at a loading concentration of 0 to 10 mM. the salt. The chromatography is carried out at a speed of 1 to 20 cm / h, preferably a superficial velocity of 4.6 cm / h. A suitable pressure can be selected to obtain the desired flow rate. Typically a pressure of 0 to 1.54 kg / cm2 (from 0 to 22 PSIG) is selected. Suitable flow rates can also be obtained by applying a negative pressure to the elution end of the column and collecting the eluent. A combination of both positive pressure and negative pressure can also be used. Any temperature can be used for the contact of the cheese processing waste stream with the anion exchange resin, as long as the temperature is not too high to cause the decomposition of the components of the sialyl oligosaccharides. Generally the ambient temperature is used from 17 to 25 ° C. When the buffer eluent is a lithium salt, the individual sialyl oligosaccharides can be isolated by concentration of the eluent to form a solid and wash the lithium salts by separating them with an organic solvent. The isolation of the lithium salt of a sialyl oligosaccharide from a lithium salt eluent is as previously described. The sodium salt of the sialyl oligosaccharides can be obtained by conventional ion exchange techniques, known to those of ordinary skill in the art. When the buffer eluent is not a lithium salt, the individual sialyl oligosaccharides can be isolated by reverse osmosis techniques.

According to a second embodiment of the present invention, a waste cheese processing stream can be treated without using an ion exchange column and without using reverse osmosis. The cheese processing waste stream such as solid lactose or an aqueous lactose solution may be in contact with a solvent, in which the sialyl oligosaccharides are extracted. The sialyl oligosaccharides which are extracted include but are not limited to 3'-sialyl lactose, 6'-sialyl lactose and 6'-sialyl lactosamine. The cheese processing waste stream may be in contact with a solvent in any suitable form to effectively extract, by solubilization, the sialyl oligosaccharides. For example, solid lactose, in the form of powder can be packed in a column and a solvent is passed through the packed column. As the solvent passes through the column, the sialyl oligosaccharides are extracted from the solid lactose. To improve the solubilization of the sialyl oligosaccharide, the solvent can be recirculated through the column, until an equilibrium concentration of the sialyl oligosaccharide in the solvent is obtained.

To improve the solubilization of the sialyl oligosaccharide, the solvent can be recirculated at elevated temperature, below the point of thermal decomposition of the sialyl oligosaccharides, preferably from 27 ° C to 80 ° C, more preferably from 60 ° C to 75 ° C to the ambient pressure. A stream of cheese processing waste may also be contacted with a solvent, such as a paste or suspension in the cheese processing waste stream in the solvent. The cheese processing waste stream is mixed with the solvent preferably in a ratio of 1: 4 v / v, more preferably 1: 3 v / v. The paste or suspension is then stirred until the sialyl oligosaccharides are solubilized in the solvent. The ratio of this waste stream from cheese processing to solvent is selected to maximize the amount of sialyl oligosaccharide recovered and minimize the amount of solvent used. Due to the high solubility of the sialyl oligosaccharides in the chosen solvent, the amount of solvent is typically much less than the volume of the cheese processing waste stream. Therefore, when the lactose is being processed, it is not necessary for the lactose to be completely dissolved.

The suspension can be stirred at any temperature, below the point of thermal decomposition of the sialyl oligosaccharides, preferably from 4 ° C to 80 ° C, more preferably from 4-27 ° C at ambient pressure. Suitable solvent systems are water, Cj ^ ^ alcohols, such as methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec-butanol, tert-butanol, tert-amyl alcohol and iso alcohol -amyl and a mixture thereof. The amount of water in the alcohol solvent system of C1_5 will vary depending on the alcohol used. Preferably the solvent contains 0-75% water (v / v), more preferably 20-70% water (v / v), more preferably 44-66% water. A particularly preferred solvent system is an aqueous ethanol solvent containing 44-66% water. When the elevated temperature is used, it is preferred to remove the solvent from the column, slurry or slurry after the maximum concentration of the sialyl oligosaccharide is reached, followed by cooling of the separated solvent. Upon cooling of the separated solvent, the solubilized lactose will crystallize and can be removed from the solvent containing the sialyl oligosaccharide, by conventional means such as filtration, centrifugation and decantation.

An aqueous solution of lactose, such as the mother liquor followed by crystallization of the lactose, can also be treated with a solvent at elevated temperature, preferably 60 to 75 ° C, more preferably 68 to 72 ° C., followed by cooling and precipitation of the lactose from the solution. The separation of the lactose precipitated from the solvent and the concentration of the solvent provides the sialyl oligosaccharide. The aqueous solution of the lactose and the solvent are mixed in a ratio of approximately 1: 3 v / v, preferably 1: 2 v / v, more preferably 1: 1 v / v. A suitable solvent for the treatment of an aqueous lactose solution is C1-5 alcohol. The separated solvent, or eluent of the column can be concentrated to produce high purity sialyl oligosaccharide. This material can be further purified by recrystallization from an aqueous ethanol and a suitable organic solvent to remove the lactose impurity. In another embodiment for the column, the paste or suspension treatment technique, a portion of the extraction solvent can be removed and passed through an anion exchange column and the solvent returned to the system. In this form, the sialyl oligosaccharide can be concentrated in an anion exchange column. The solvent that is passed through the anion exchange resin can be removed continuously or discontinuously. Once the anion exchange column has been saturated with sialyl oligosaccharide, the column can be removed from the system and purged to obtain the sialyl oligosaccharide. A suitable purge solution is 120 mM LiOAc. Generally from 2 to 5, preferably 4 volumes of the column of the purge solution are used to remove the negatively charged materials from the anion exchange resin, made at room temperature. Suitable anion exchange resins, contact conditions and purge conditions have been previously described in the foregoing. Sialyloligosaccharides can also be extracted from waste streams of the serum using supercritical CO2 extraction techniques, in a method analogous to the methods used to extract caffeine from coffee beans. A technique for extracting caffeine from coffee beans using supercritical, wet C02 is described in U.S. Patent Nos. 3,806,619 and 4,260,639. In general, the supercritical C02 extraction method comprises contacting the lactose in an aqueous solution of lactose with supercritical C02, under conditions to effect the solubilization of the sialyl oligosaccharides by supercritical C02. The supercritical C02 containing sialyl oligosaccharides is separated from the lactose or the aqueous solution of lactose, then the C02 is removed by evaporation, leaving behind the extracted sialyl oligosaccharides. Other features of the invention will become apparent in the course of the following description of exemplary embodiments, which are given for illustration of the invention and are not intended to be limiting thereof. Example 1 227 kg (500 pounds) of edible lactose (available from Land O'Lakes Dairy) are dissolved in 2,000 1 of water at about room temperature. The particular material is removed by passing the solution through a 5 μm filter. The solution is passed over 15 1 of DOWEX 50 x 8 cation exchange resin. The eluent is passed over 15 1 of the DOWEX 1 x 8 anion exchange resin. The resin is then washed with water to remove any residual lactose, then with 10 volumes of the 50 mM NaOAc column. The NaOAc eluent is concentrated and diafiltered with 6 volumes of the water column by reverse osmosis to desalt (using an exclusion membrane of 400 Dalton units at 28.12 kg / cm2 (400 PSIG) .10 1 of a solution containing 55 is obtained. grams of the sialyl oligosaccharide.

Example 2 The solution of Example 1 is loaded onto a 76 x 36 cm column containing 45 1 of DOWEX 1x2 resin and eluted with 190 1 of 120 mM NaOAc, at a rate of 55 ml / minute. 6'-Silyl-lactose eluted after 40 to 47 h, while 3'-sialyl-lactose eluted after 48 to 55 h. The fractions containing the specific sialyl oligosaccharides were adjusted to a pH of 4.8-5.3 +/- 0.1, then concentrated and desalted by diafiltration with 10 volumes of the water column. Then the fractions are adjusted to a pH of 8.25. The fractions are concentrated to approximately 200 mg / ml by rotary evaporation, passed through a 0.2 μm filter and diluted with four volumes of EtOH and 13.3 volumes of acetone under vigorous stirring to form a suspension. The suspension is cooled to about 4 ° C and the solids are isolated by centrifugation, followed by washing with acetone. Example 3 Lactose columns of 1 pass 5 g of lactose (from Land O'Lakes Dairy) containing approximately 1.5 mg of 3'-sialyl lactose are placed on a column and washed with 10 ml of solvent, either aqueous EtOH at 66% or 45% aqueous EtOH either at 4 ° C or at room temperature (TA). Washing is analyzed for the percentage of 3 'sialyl-lactose which is eliminated by CLAP using peak integration. Conditions% Yield of 3 '-SL 45% TA 6.26 45% 4 ° C 3.7 66% TA 2 66% 4 ° C 1.4 Example 4 Column of 5 a lactose with recirculation 5 g of lactose (from Land O'Lakes Dairy ) containing approximately 1.5 mg of 3'-sialyl lactose are placed in a column and washed with a wash recirculation pump (66% EtOH / 4 ° C). After 14 h, the washing is analyzed. Conditions% of Performance of 3 '-SL Circ. 66% 4 ° C 17% Example 5 Pasta Extractions 5 g of lactose (from Land O'Lakes Dairy) containing approximately 1.5 mg of 3'-sialyl lactose are placed in a flask and stirred in 20 ml of solvent, either 66% aqueous EtOH or 45% aqueous EtOH either at 4 ° C or Temperature Environment (TA). Lactose is never obtained in solution, but the 3'-sialyl-lactose dissolved. The supernatant was analyzed. Conditions% Yield of 3 '-SL 45% TA 33 45% 4 ° C 9.6 66% TA 18 66% 4 ° C 22.3 Example 6 Heat Extraction 5 g of lactose (from Land O'Lakes Dairy) containing approximately 1.5 mg of 3'-sialyl lactose is placed in a flask and heated to obtain the lactose in 150 ml of 66% EtOH, then cooled with an additional 100 ml of 66% EtOH at RT and stirred overnight 4 ° C. A precipitate is rapidly crushed from the solution. The supernatant was analyzed: Conditions% Yield of 3 '-SL hot / cold EtOH 66% 70.5 Example 7 Extraction with Heat - Variable Conditions 6.4 g of lactose (from Land O'Lakes Dairy) (~ 1.92 mg of 3' - SL) is heated to 70 ° C to obtain a solution of 20 ml of water. Then the concentrated lactose solution is stirred for 16 hours either at 4 ° C or at room temperature (RT) and various concentrations of EtOH to precipitate the lactose and retain the 3'-SL. Conditions% of Performance of 3 '-SL 0% EtOH at RT 100 33% EtOH at RT 32 50% EtOH at RT 36 66% EtOH at RT ~ 7 0% EtOH at 4 ° C 63 33% EtOH at 4 ° C 50 50% EtOH at 4 ° C 26 66% EtOH at 4 ° C 24 Example 8 Heat Extraction - Variable Conditions with Greater Contact Time 60 g of lactose (from Land O'Lakes Dairy) (~ 18 mg of 3'-SL) are dissolved in 100 ral of H20 by heating at 70 ° C for about 10 minutes. Aliquots of equal size are placed under various conditions and stirred for approximately 36 g. The samples were analyzed by CLAP. Conditions% of Performance of 3 '-SL 0% EtOH at RT 100 33% EtOH at RT 18.3 50% EtOH at RT 8.3 66% EtOH at RT 8.7 0% EtOH at 4 ° C 100 33% EtOH at 4 ° C 27.6 50% EtOH at 4 ° C 15.2 66% EtOH at 4 ° C 100 Example 9 Mother liquor - Heat experiment 100 ml of mother liquor are heated at 70 ° C for approximately 10 minutes, then cooled to 4 ° C with a 2x volume of 95% EtOH. Allow to stir for 36 hours. The sample is analyzed in CLAP for the content of 3'-SL. Conditions% Yield of 3 '-SL ML of 0% EtOH at 4 ° C 33 * * * * * * * * * Obviously numerous modifications and variations of the present invention are possible in light of the above teachings. Therefore, it should be understood that within the scope of the appended claims, the invention may be practiced in any other way than that specifically described herein.

Claims (31)

1. CLAIMS 1. A method for processing the cheese processing waste stream, characterized in that it comprises: i) contacting a cheese processing waste stream with an anion exchange resin; ii) removing the anion exchange resin from the cheese processing waste stream and eluting the anion exchange resin with an aqueous solution of a lithium salt, to produce an eluent; iii) concentrating the eluent to form a solid comprising a lithium salt and a lithium salt of a cheese processing waste; and iv) washing the solid with an organic solvent, dissolving the lithium salt and leaving the lithium salt of a waste from processing the cheese as a solid. The method according to claim 1, characterized in that the cheese processing waste stream is fluid cheese whey. The method according to claim 1, characterized in that the cheese processing waste stream is the solids obtained by crystallization of the lactose from the cheese whey. 4. The method according to claim 1, characterized in that the cheese processing waste stream is the mother liquor obtained by crystallization of the lactose from the cheese whey. The method according to claim 1, characterized in that the cheese processing waste stream is selected from the group consisting of colostrum, milk, milk powder, whole serum, permeated demineralized serum, regeneration stream of demineralized serum permeate, serum permeate, crystallized lactose, spray dried lactose, whey powder, edible lactose, lactose, refined lactose and USP lactose. 6. The method of compliance with the claim 1, characterized in that the lithium salt is selected from the group consisting of lithium acetate, lithium carbonate, lithium sulfate, lithium perchlorate, lithium chloride, lithium bromide and a mixture of the same. 7. The method of compliance with the claim 1, characterized in that the organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, 3-pentanone, diethyl ether, t-butyl methyl ether and a mixture thereof. 8. The method in accordance with the claim I, further characterized in that it comprises treating the cheese processing waste stream to remove positively charged materials, before contacting the anion exchange resin. 9. The method according to claim 8, characterized in that the treatment is with a cation exchange resin. 10. The method according to claim 1, characterized in that the lithium salt is lithium acetate. 11. A method of processing the waste stream from cheese processing, characterized in that it comprises: i) contacting the waste stream processing the cheese with a solvent and extracting a sialyl oligosaccharide with the solvent; ii) separating a solvent-containing sialyl oligosaccharide from the cheese processing waste stream; and iii) isolating a sialyl oligosaccharide from the solvent containing sialyl oligosaccharide. 12. The process in accordance with the claim II, characterized in that the solvent is selected from the group consisting of water, C1-5 alcohol and a mixture thereof. 13. The process according to claim 11, characterized in that the waste stream processing the cheese is lactose. The process according to claim 11, characterized in that the cheese processing waste stream is the mother liquor obtained by crystallization of the lactose from the cheese whey. 15. The process in accordance with the claim 11, characterized in that the solvent is aqueous ethanol at 44-66% (v / v). 16. The process according to claim 11, characterized in that the solvent is brought into contact with the cheese processing waste stream at a temperature of 20 to 80 ° C. 17. The process according to claim 11, characterized in that the solvent is brought into contact with the waste stream processing the cheese at a temperature of 40 to 45 ° C. 18. The process according to claim 11, characterized in that the solvent is separated from the cheese processing waste stream at a temperature of 20 to 80 ° C. 19. The process according to claim 11, characterized in that the solvent is separated from the processing waste stream of the cheese at a temperature of 40 to 45 ° C. 20. The process in accordance with the claim 11, characterized in that the contact is carried out as a suspension of the cheese and solvent processing waste stream. 21. The process according to claim 14, characterized in that the contact is carried out in a mother liquor at a solvent ratio of 1: 2 (v / v). 22. The process according to claim 14, characterized in that the contact is carried out at a temperature of 60 to 75 ° C. 23. The method according to the claim I, characterized in that the lithium salt of the cheese processing waste is a sialyl oligosaccharide. The method according to claim 23, characterized in that the sialyl oligosaccharide is selected from the group consisting of 3 '-sialylactose, 6'-sialyl lactose and 6'-sialyl lactosamine and a mixture thereof. 25. The method of compliance with the claim II, characterized in that the sialyl oligosaccharide is selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose and 6'-sialyl lactosamine and a mixture thereof. 26. The method according to claim 11, characterized in that the sialyl oligosaccharide is 3'-sialyl lactose. 27. The method according to claim 11, characterized in that the sialyl oligosaccharide is 6'-sialyl-lactoea. 28. The method according to claim 11, characterized in that the sialyl oligosaccharide is 6'-sialyl-lactosamine. 29. The method according to claim 23, characterized in that the sialyl oligosaccharide is 3'-sialylactose. 30. The method of compliance with the claim 23, characterized in that the sialyl oligosaccharide is 6'-sialyl lactose. 31. The method according to claim 23, characterized in that the sialyl oligosaccharide is 6'-sialyl-lactosamine.